This Program Project on Structure-Based Antagonism of HlV-1 Envelope Function in Cell Entry will lead to a new understanding of the HlV-1 envelope (Env) structure and structural transitions that accompany cell entry, that in turn will permit the development of potent, selective antagonists of HIV-1 entry for both intervention and prevention of the AIDS pandemic. The major focus of the Synthetic Thrust (Project 3) within this Program Project will be the development of new small molecule antagonists of the CD4-gp120 protein-protein interaction that mediate viral entry into target host cells. The CD4 binding site of gp120 has great promise as a major therapeutic target site on the Env protein for inhibitor design, given the well-defined binding surface that is highly conserved in diverse virus sub-types. Ligand binding at this site, however, has also evolved to trigger the allosteric activation process that the virus employs to mediate fusion. As we proceed forward, we will build upon our recent discovery of DMJ-ll-121, a small molecule that targets two highly conserved sites of gp120 and in turn neutralizes HlV-1 without triggering the allosteric response in gp120. The guidelines developed during the design of DMJ-ll-121 provide a roadmap for the discovery of new small molecule inhibitors of the dynamic protein-protein interactions. This roadmap will also be employed to optimize new leads identified by the screening programs within the Program Project. The Synthetic Thrust, in conjunction with the other Projects will also prepare conjugates of the antagonists with molecular probes to study structurally the biochemical mechanisms associated with HlV-1 neutralization. Photoaffinity labels will be attached covalently to highly active antagonists to elucidate the binding sites of inhibitors that entrap the Env in an inactive conformation. The dynamic processes associated with small molecule binding to the HIV-1 Env trimer will also be interrogated with recently developed single molecule FRET probes. Finally, innovative covalent labeling strategies to append a tetrairidium cluster to validated antagonists will facilitate binding site analysis within solubilized Env trimers by SP-cryoEM.